Elevated blood cholesterol is a major risk factor for heart disease, which is responsible for ⅓ of all deaths worldwide. Phytosterols have been found effective in lowering elevated cholesterol when incorporated into a variety of low fat foods, demonstrating an 18 to 15% reduction in LDL cholesterol with no reported adverse health effects. Phytosterols have been shown to inhibit uptake from the intestine of dietary and endogenously-produced cholesterol (see, for example, Moreau, et al. (2002) Prog. Lipid Res. 41:457-500).
Naturally occurring phytosterols include, and are not limited to, isofucosterol, sitosterol, stigmasterol, campesterol, cholesterol, cycloartenol, and dihydrobrassicasterol. The most abundant sterols commonly are campesterol, sitosterol, and stigmasterol. Phytosterols commonly occur as free alcohols, or as fatty acid esters, steryl glycosides, or acylated steryl glycosides which are commonly referred to as phytosterol conjugates. Phytostanols are fully-saturated phytosterols (contain no double bonds) and, as such, are considered a subgroup of phytosterols. Phytostanols occur in trace levels in many plant species. Phytosterols can be converted to phytostanols by chemical hydrogenation.
Products and compositions comprising phytosterols are available in the United States for the purpose of increasing heart health. There is a demand for phytosterol formulations that could be included in beverages, dairy drinks, and non-fat foods. Thus, plants having altered phytosterol compositions may be useful in the preparation of the above-mentioned compositions and the compositions will have a different distribution of phytosterols.
Phytosterols, including phytostanols, and triterpenes are biosynthesized via the isoprenoid pathway. In this pathway, two molecules of farnesyl pyrophosphate are joined head-to-head to form squalene, a triterpene. Squalene is then converted to 2,3-oxidosqualene. Various oxidosqualene cyclases catalyze the cyclization of 2,3-oxidosqualene to form various polycyclic skeletons including, but not limited to cycloartenol, lanosterol, lupeol, isomultiflorenol, β-amyrin, α-amyrin, and thalianol. This cyclization event catalyzed by oxidosqualene cyclases forms a branch point between the sterol and triterpene saponin biosynthetic pathways. The various oxidosqualene cyclases are evolutionarily related (Kushiro, T., et al. (1998) Eur. J. Biochem. 256:238-244) and produce a wide variety of three-, four-, and five-ring structures that can be further modified.
For sterol synthesis, the cyclization of 2,3-oxidosqualene is catalyzed by the 2,3-oxidosqualene cyclases, cycloartenol synthase and lanosterol synthase. Cycloartenol (in photosynthetic organisms) and lanosterol (in non-photosynthetic organisms) are 30 carbon, 4-ring structures that can be further modified to form sterols. In photosynthetic organisms, sterols have a wide range of functions including regulation of membrane fluidity and as precursors for the brassinosteroids. In some plants, sterols can also be glycosylated to form steroidal saponins. Cycloatenol serves as a precursor for the production of numerous other sterols. In most plants, cycloartenol is converted to 24-methylene cycloartenol, cycloeucalenol, obtusifoliol, isofucosterol, sitostero, stigmasterol, campesterol, and cholesterol. Cycloartenol is formed by the enzyme cycloartenol synthase (EC 5.4.99.8), also called 2,3-epoxysqualene-cycloartenol cyclase. The basic nucleus of cycloartenol can be further modified by reactions such as desaturation or demethylation to form the common sterol backbones.
For triterpene saponin synthesis, the cyclization of 2,3-oxidosqualene is catalyzed by 2,3-oxidosqualene cyclases, such as lupeol synthase, β-amyrin synthase, α-amyrin synthase, isomultiflorenol synthase, thalianol synthase and dammarenediol synthase. Lupeol, β-amyrin, α-amyrin, isomultiflorenol and thalianol, can be further modified (e.g., oxidation, substitution, and glycosylation) to form triterpene saponins. For example, the basic β-amyrin ring structure may be modified by glycosylation (sometimes preceded by hydroxylation) to form triterpene saponins. The function of triterpene saponins is unclear although it is thought that they play a defense role against pathogens in plant tissues.